Huo-Shu H. Houng

Differing Influences of Virus Burden and Immune Activation on Disease Severity in Secondary Dengue-3 Virus Infections

Dengue hemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage, thrombocytopenia, and hepatic inflammation. The interrelationships among virus burden, immune activation, and development of DHF were examined in 54 children with secondary dengue-3 virus infections participating in a prospective, hospital-based study. DHF was associated with higher mean plasma viremia early in illness and earlier peak plasma interferon-gamma levels. Maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia. Maximum plasma levels of interleukin (IL)-10 and soluble tumor necrosis factor receptor-II correlated with the degree of thrombocytopenia, independently of viremia levels. Hepatic transaminase elevation correlated with plasma soluble IL-2 receptor levels and not with viremia levels. Quantitative differences in virus burden and host immune responses, and the timing of type 1 cytokine responses, have differing influences on the severity of disease manifestations during secondary dengue-3 virus infections.

Detection of Shigella by a PCR Assay Targeting the ipaH Gene Suggests Increased Prevalence of Shigellosis in Nha Trang, Vietnam

ABSTRACT Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.

Outbreak of Febrile Respiratory Illness Associated with Adenovirus 11a Infection in a Singapore Military Training Camp

ABSTRACT Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China.

A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive escherichia coli in human feces

A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and enteroinvasive Escherichia coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipaH and ial, cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100-200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., < 10(4) CFU/gram. Serotype-specific primers derived from the rfc genes of differentiate among Shigella serotypes in the laboratory, such as S. sonnei, S. flexneri, and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc-specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.

Development of a ceuE-based multiplex polymerase chain reaction (PCR) assay for direct detection and differentiation of Campylobacter jejuni and Campylobacter coli in Thailand

A novel ceuE-based multiplex PCR system was developed as an efficient diagnostics test to detect and differentiate C. jejuni and C. coli. There is no cross reactivity between C. jejuni and C. coli. In addition, the assay does not produce a positive signal from other enteric bacteria including Salmonella, Shigella and Escherichia coli strains. Campylobacter detection sensitivity was determined to be equivalent to previously reported PCR for other enteric bacteria. We also noticed that silicon dioxide extraction can improve Campylobacter detection sensitivity from infected stool samples. It was demonstrated that the PCR assay developed in this study had a much better Campylobacter detection rate than the traditional culturing method (77% versus 56%). However, we also identified small numbers of culture positive stools (8%, or 16 out of 202 samples) that did not yield PCR positive results for Campylobacter. These PCR negative/culture positive stools were proven to be inhibitory to PCR amplification.

Genome sequences of Human Adenovirus 14 isolates from mild respiratory cases and a fatal pneumonia, isolated during 2006-2007 epidemics in North America

BackgroundHuman adenovirus 14 (HAdV-14) is a recognized causative agent of epidemic febrile respiratory illness (FRI). Last reported in Eurasia in 1963, this virus has since been conspicuously absent in broad surveys, and was never isolated in North America despite inclusion of specific tests for this serotype in surveillance methods. In 2006 and 2007, this virus suddenly emerged in North America, causing high attack rate epidemics of FRI and, in some cases, severe pneumonias and occasional fatalities. Some outbreaks have been relatively mild, with low rates of progression beyond uncomplicated FRI, while other outbreaks have involved high rates of more serious outcomes.Methodology and FindingsIn this paper we present the complete genomic sequence of this emerging pathogen, and compare genomic sequences of isolates from both mild and severe outbreaks. We also compare the genome sequences of the recent isolates with those of the prototype HAdV-14 that circulated in Eurasia 30 years ago and the closely related sequence of HAdV-11a, which has been circulating in southeast Asia.ConclusionsThe data suggest that the currently circulating strain of HAdV-14 is closely related to the historically recognized prototype throughout its genome, though it does display a couple of potentially functional mutations in the fiber knob and E1A genes. There are no polymorphisms that suggest an obvious explanation for the divergence in severity between outbreak events, suggesting that differences in outcome are more likely environmental or host determined rather than viral genetics.

Emergent 2009 influenza A(H1N1) viruses containing HA D222N mutation associated with severe clinical outcomes in the Americas

BACKGROUND During the 2010-2011 influenza season, a small sub-group of 2009 influenza A(H1N1) viruses (hereafter referred to as 2009 A(H1N1)) emerged that was associated with more severe clinical outcomes in Ecuador and North America. Genetically, the haemagglutinin (HA) of this sub-clade was distinct from HAs found in viruses associated with severe outbreaks in 2010 from the United Kingdom and from other global specimens isolated earlier in the season. OBJECTIVE We report the emergence of a novel 2009 A(H1N1) variant possessing a re-emergent HA D222N mutation obtained from patients with severe respiratory illnesses and phylogenetically characterise these D222N mutants with other severe disease-causing variants clustering within a common emerging sub-clade. CASE REPORTS In early 2011, three cases of 2009 A(H1N1) infection, two from Quito, Ecuador, and one from Washington, DC, USA, were complicated by severe pneumonia requiring mechanical ventilation, resulting in one fatality. These cases were selected due to the reported nature of the acute respiratory distress (ARD) that were captured in Department of Defence (DoD)-sponsored global influenza surveillance nets. RESULTS Genetically, the 2009 A(H1N1) strains isolated from two of the three severe cases carried a prominent amino acid change at position 222 (D222N) within the primary HA receptor binding site. Furthermore, these cases represent an emerging sub-clade of viruses defined by amino acid changes within HA: N31D, S162N, A186T and V272I. Phylogenetically, these viruses share a high degree of homology with strains associated with recent fatal cases in Chihuahua, Mexico. DISCUSSION Previously, enhanced virulence associated with the change, D222G, has been clinically linked to severe morbidity and mortality. Initial observations of the prevalence of a novel sub-clade of strains in the Americas suggest that viruses with a re-emergent D222N mutation may too correlate with severe clinical manifestations. These findings warrant heightened vigilance for emerging sub-clades of 2009 A(H1N1) and presumptive clinical implications.

Diagnostic Approach to Acute Diarrheal Illness in a Military Population on Training Exercises in Thailand, a Region of Campylobacter Hyperendemicity

ABSTRACT High rates of Campylobacter fluoroquinolone resistance highlight the need to evaluate diagnostic strategies that can be used to assist with clinical management. Diagnostic tests were evaluated with U.S. soldiers presenting with acute diarrhea during deployment in Thailand. The results of bedside and field laboratory diagnostic tests were compared to stool microbiology findings for 182 enrolled patients. Campylobacter jejuni was isolated from 62% of the cases. Clinical and laboratory findings at the time of presentation were evaluated to determine their impact on the posttest probability, defined as the likelihood of a diagnosis of Campylobacter infection. Clinical findings, the results of tests for inflammation (stool occult blood testing [Hemoccult], fecal leukocytes, fecal lactoferrin, plasma C-reactive protein), and the numbers of Campylobacter-specific antibody-secreting cells in peripheral blood failed to increase the posttest probability above 90% in this setting of Campylobacter hyperendemicity when these findings were present. Positive results by a Campylobacter-specific commercial enzyme immunoassay (EIA) and, less so, a research PCR were strong positive predictors. The negative predictive value for ruling out Campylobacter infection, defined as a posttest probability of less than 10%, was similarly observed with these Campylobacter-specific stool-based tests as well the fecal leukocyte test. Compared to the other tests evaluated, the Campylobacter EIA is a sensitive and specific rapid diagnostic test that may assist with diagnostic evaluation, with consideration of the epidemiological setting, logistics, and cost.

Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

ABSTRACT Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

Adenovirus microsatellite reveals dynamics of transmission during a recent epidemic of human adenovirus serotype 14 infection.

ABSTRACT This study reveals diverse-length polymorphisms in long mononucleotide repeats (microsatellites) in several serotypes of epidemic human respiratory adenovirus. The length of one of these microsatellites, a homopolymeric thymidine [poly(T)] repeat, is measured in 68 isolates of adenovirus serotype 14. These isolates were collected during a series of sudden and sometimes fatal outbreaks among both military recruits and civilians as the virus emerged for the first time in the United States in 2006 and 2007. The results demonstrate the usefulness of adenoviral microsatellites as high-resolution molecular strain markers. The described homopolymer is hypervariable in length, varying from 12 to 17 bp in the analyzed sample set. All intermediate lengths were identified in at least one isolate. Furthermore, the specific length of the marker is stable for significant periods of time (up to 7 months) at individual sites where the virus is in consistent circulation. The microsatellite also can maintain specific length identity through site-to-site transmission events, as determined by the analysis of isolates from three advanced training sites that appeared to be subject to pathogen transfer from one of the affected recruit training installations. Public database searches revealed that the polymorphic nature of the microsatellite extends to other species B serotypes, and that other polymorphic microsatellites can be identified readily in a variety of epidemic respiratory adenovirus clades. This study shows that microsatellites are a ubiquitous source of polymorphic markers for human adenoviruses and demonstrates their use through an epidemiological analysis of isolates from a recent North American epidemic.